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This review has attempted to integrate three areas of cellular bioenergetics to present a novel and comprehensive view of heart high-energy phosphate metabolism. The goal has been to provide a rational view for the functions of phosphocreatine, creatine, and creatine kinase in the energy metabolism of muscle. The first point is that mitochondrial respiratory control is influenced by changes in the concentration of ADP, stimulating the adenine nucleotide translocase and oxidative phosphorylation. Secondly, as a consequence of the proximity of mitochondrial creatine kinase to the translocase, there appears to be a kinetic preference for ADP generated by the forward creatine kinase reaction. As a result, in heart, it can be viewed that the end product of oxidative phosphorylation is phosphocreatine. Finally, thermodynamic considerations suggest that phosphocreatine plays a major role to maintain or buffer the ATP content of the myocardium. Under conditions of increased ATP turnover, large-scale increases in the concentration of ADP, along with major decreases in ATP, are minimized by the creatine kinase equilibrium. The system responds to such a demand with substantial changes in phosphocreatine and creatine, which can kinetically increase the rate of mitochondrial creatine kinase and thus oxidative phosphorylation. Theoretical enzymologists have long argued whether enzymes are under kinetic or thermodynamic control. Heart creatine kinase may be a unique example where both types of control simultaneously operate in different microenvironments, with mitochondrial creatine kinase kinetically controlled, while the sarcoplasmic isozyme is influenced by equilibrium thermodynamics. Overall, heart creatine kinase may be a unique example of "kineto-dynamic" metabolic integration.

This paper summarizes the data concerning the role of the creatine phosphokinase system in muscle cells with main attention to the cardiac muscle. Creatine phosphokinase isoenzymes play a key role in the intracellular energy transport from mitochondria to myofibrils and other sites of energy utilization. Due to the existence of the creatine phosphate pathway for energy transport, intracellular creatine phosphate concentration is apparently an important regulatory factor for muscle contraction which influences the contractile force by determining the rate of regeneration of ATP directly available for myosin ATPase, and at the same time controls the activator calcium entry into the myoplasm across the surface membrane of the cells.

Several recent studies have demonstrated the presence of creatine kinase and of phosphorylcreatine in a variety of cells besides striated muscle and brain cells. The total creatine kinase and phosphagen levels in these cells encompass a wide range of values. The available data are collected in this article to demonstrate that the variation of the enzyme and phosphagen concentrations is not random but that the two are interrelated. With both the major isoenzymes of creatine kinase, namely the muscle type and the brain type, the basal levels of phosphorylcreatine follow closely the cellular creatine kinase levels. A hypothesis is presented in which the enzyme itself is the major determinant of phosphorylcreatine content by virtue of its ability to act as an intracellular binding protein for creatine derived from extracellular fluid, and also for cellular ADP. The proposed mechanism further predicts that in cells that contain high levels of actin and thus sequester the cytoplasmic free ADP (e.g. most muscle cells), a high level of creatine kinase can effectively regulate the myokinase reaction by its ability to bind ADP. The net effect of such regulation is to conserve the adenine nucleotide pools in the cell. The evolutionary advantage of these two regulatory functions of creatine kinase in terms of energy conservation is discussed.

The method of the penetrating ions allows to observe the creatine phosphokinase function of heart mitochondria in steady state conditions. The addition of ADP to respiring mitochondria decreases the membrane potential in the course of the ATP synthesis. The subsequent addition of creatine decreases the membrane potential for a long period of time because of ADP formation in creatine phosphokinase reaction. It was shown in the experiments with maintainance of the constant concentration of ADP or ATP in the medium that the ADP concentration in the medium acts mainly on the membrane potential during the creatine phosphate synthesis. There is no effect of creatine on the membrane potential of liver mitochondria which have no creatine phosphokinase.

It has been shown that the contents of mitochondria and mitochondrial isoenzyme of creatine phosphokinase are almost identical in skeletal and heart muscles. In mitochondria from both types of muscle creatine phosphokinase is functionally coupled to ATP-ADP translocase. This kind of coupling ensures complete conversion of mitochondrial ATP energy into the energy of creatine phosphate and effective control of oxidative phosphorylation by the creatine phosphokinase reaction. It has also been shown that all isoenzymes of creatine phosphokinase from heart and skeletal muscle have very similar kinetic properties. Significant differences have been found to exist between isoenzyme patterns of these muscles and also in distribution of different isoenzymes in the cells. In skeletal muscle cells creatine phosphokinase is present mainly as cytosolic MM isoenzyme; about 6% of total cellular activity is localised also in mitochondria. Due to high activity of cytosolic isoenzyme the total activity of creatine phosphokinase is about three times higher in skeletal muscle than in cardiac muscle. It has been also shown that phosphoenolpyruvate and glucose-6-phosphate do not have any inhibitory effect on creatine phosphokinase.

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.

Creatine phosphate (10 and 20 mM) increased both the force of contraction and action potential duration in isolated frog heart strips. In concentration of 75 mM creatine phosphate increased the force of contraction during first minutes with its following decrease. Action potential duration was also increased during this period. These data suggest existence of receptory sites for creatine phosphate in the heart cells: the myofibrillar creatine phosphokinase system where creatine phosphate increases the force of contraction; and the cellular membranes where creatine phosphate decreases the rate of membrane repolarization. Dinitrofluorobenzene (1.5 mM) completely inhibited contraction of the heart strips and significantly decreased the duration and intensity of action potential. The data obtained are discussed from the view--point of existence of a creatine phosphate pathway for intracellular energy transport in the heart cells.

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.

Glycerinated rabbit psoas muscle fibers containing native CPK, ATPase, and myokinase activities were used and isometric contraction and relaxation responses to either ADP or ATP + CP or to ATP alone in the presence and absence of P1, P5-di(adenosine-5'-pentaphosphate), a myokinase inhibitor, were compared. In previous (14) work it was shown that CP generated more efficient and faster contraction and relaxation of glycerinated muscle fibers than ATP. The present work deals with the role of myokinase in the differential response of fibers to CP and ATP. Inhibition of the myokinase activity of these fibers caused slight diminution of the rate of contraction at physiological concentrations of ATP. Uninhibited fibers were not able to reach maximum contraction, because the tension began to drop gradually even in the presence of Ca2+. Addition of Ap5A permitted maximum contraction and the ability to stay at the contracted state. In the case of CP + adenosine nucleotides (ATP or ADP), myokinase activity decreased the rate of tension development which was statistically significant after 5-7 sec of contraction. Thus, a higher tension was obtainable when myokinase was inhibited. At high concentration of adenine nucleotides (greater than 2 mM) and in the absence of Ap5A, not only the maximum tension never was reached, but a spontaneous drop in tension was observed before addition of EGTA, as was seen with ATP alone. Relaxation was faster and more complete in the presence of uninhibited myokinase activity except that the ADP was low (125 mM). These observations provide further evidence for a close functional interaction of these three enzymes in the mechanism of contraction and relaxation, giving further support to the notion of the creatine-phosphocreatine energy shuttle.

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.

Development proceeds by way of a discrete yet overlapping series of biosynthetic and restructuring events that result in the continued molding of tissues and organs into highly restricted and specialized states required for adult function. Individual molecules and cells are replaced by molecular and cellular variants, called isoforms; these arise and function during embryonic development or later life. Isoforms, whether molecular or cellular, have been identified by their structural differences, which allow separation and characterization of each variant. These isoforms play a central and controlling role in the continued and dynamic remodeling that takes place during development. Descriptions of the individual phases of the orderly replacement of one isoform for another provides an experimental context in which the process of development can be better understood.

The review contains the analysis of present-day concepts on the physiological role of the creatine kinase system. A hypothesis on the buffering functions of the creatine kinase system which ensures a constant ATP level in cells and a hypothesis according to which phosphocreatine is a macroergic phosphate carrier from mitochondria to the sites of their utilization are considered. In connection with the creatine phosphate carrier hypothesis according to which the transport function of the creatine kinase system is provided for by an effective function of mitochondrial creatine kinase, feasible mechanisms of mitochondrial creatine kinase activity regulation are considered: as a result of creation of local concentration of nucleotide substrates as well as changes in the properties of creatine kinase itself which may result from the enzyme conversion from the membrane-bound to the free form or from the interconversion of oligomeric forms of the enzyme.

In the absence of creatine phosphate, MgATP produced relaxation of rigor tension in chemically-skinned right papillary muscles of the rat, the half maximal effect being obtained at 1.8 mM MgATP. In the presence of 12 mM creatine phosphate and 250 microM ADP, a decrease in MgATP concentration even to 10(-9) M never induced rigor tension. At a very low MgATP concentration (10(-6) M), the half maximal relaxing effect was obtained with 2 mM creatine phosphate, a value close to the Km of isolated MM-creatine kinase for this substrate, or with 14 microM MgADP, a value 5 times lower than the reported Km. An exogenous MgATP regenerating system (phosphoenol pyruvate + pyruvate kinase) was not able to fully relax the fibres. When MM-creatine kinase was inhibited by fluorodinitrobenzene, the dependency of rigor tension on MgATP became the same as it was without creatine phosphate. After washing out the fluorodinitrobenzene the addition of exogenous MM-creatine kinase for half an hour fully relaxed rigor tension; moreover, this effect persisted even after prolonged washout. These results show that endogenous MM-creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a localized high MgATP/MgADP ratio; they also suggest the existence of rapidly exchangeable binding sites for MM-creatine kinase in cardiac myofibrils.

BC3H1 is a cell line that undergoes a musclelike pattern of differentiation under the appropriate conditions. We have examined the control of the synthesis of proteins characteristic of differentiated muscle in these cells as a function of their position in the cell cycle. We define two positions in the cell cycle where BC3H1 cells can remain stably quiescent. G1d is a restriction point early in the G1 portion of the cell cycle that permits the synthesis of muscle-specific proteins and is probably identical to G0. The second restriction point, G1q, occurs approximately 4 hr later in the G1 portion of the cell cycle and does not permit the synthesis of muscle-specific proteins. Movement of the cells from G1d to G1q occurs when fibroblast growth factor is added to the cells and is reversed when this growth factor is removed. Repression of the synthesis of muscle-specific proteins occurs when fibroblast growth factor is added to cells in G1d. In the case of the muscle form of creatine phosphokinase (M-CPK), the decline in the rate synthesis of this protein is a consequence of a decreased level of its mRNA. By contrast, the repression of alpha-actin synthesis, a protein synthesized only in differentiated cells, appears to be controlled at the translational level. The effect of fibroblast growth factor and other mitogens in these cells require activation of tyrosine kinase(s), but the intracellular targets of these kinases are not known. Studies by others suggest that activation of the ras oncogene can mimic the action of mitogenic polypeptides on these and other muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The control exerted by the adenine nucleotide translocator and the ADP-regenerating system on oxidative phosphorylation was studied in isolated rat-liver mitochondria respiring with succinate. At intermediate rates of respiration the flux control coefficient (control strength) of the adenine nucleotide translocator on respiration was much higher with creatine/creatine kinase than with glucose/hexokinase as the ADP-regenerating system. In contrast, at the same rate of respiration the flux control coefficient of creatine kinase was much lower than that of hexokinase. On addition of a small amount of carboxyatractyloside to mitochondria respiring in the presence of glucose/hexokinase or creatine/creatine kinase, the rate of respiration decreased abruptly and then increased again to a new steady state which was lower with creatine/creatine kinase than with glucose/hexokinase. In the new steady state, the extramitochondrial ATP/ADP ratio was lower with glucose/hexokinase than with creatine/creatine kinase. At the same rate of respiration, the elasticity coefficient of creatine kinase towards the extramitochondrial ATP/ADP ratio was much higher than that of hexokinase. The connectivity theorem [Kacser, H. and Burns, J.A. (1973) in Rate Control of Biological Processes (Davies, D.D., ed.) pp. 65-104, Cambridge University Press, London] which relates the flux control coefficients of two adjacent enzymes to their elasticity coefficients towards the common intermediate, provides an explanation for the difference in flux control coefficient of the adenine nucleotide translocator with the two ADP-regenerating systems. Using principles developed by R. Heinrich and T.A. Rapoport [BioSystems 7, 130-136 (1975)], the flux control coefficients of the adenine nucleotide translocator and hexokinase on phosphorylation were also calculated from the elasticity coefficients of these enzymes towards the extramitochondrial ATP/ADP ratio and the controls exerted by these enzymes on the extramitochondrial ATP/ADP ratio. The calculated values were approximately 30% lower than the values measured directly. The elasticity coefficient of the adenine nucleotide translocator towards the extramitochondrial ATP/ADP ratio was determined, using either the connectivity theorem of Kacser and Burns (loc. cit.) or a new connectivity theorem developed by Westerhoff (following paper in this journal). Good agreement was obtained using the different methods of calculation.(ABSTRACT TRUNCATED AT 400 WORDS)